Opioids are a mainstay of treatment for acute and chronic pain. Unfortunately, chronic opioid use is highly problematic in large part due to the loss of treatment effect over time and the need to escalate doses. Strong preliminary and published evidence suggests that pain signaling and opioid effects converge on BDNF expression in the dorsal horn of the spinal cord to accelerate tolerance, paradoxically enhance pain and worsen functional outcomes after acute injuries including surgery and when treating chronic disease. Epigenetic effects appear to play a prominent role. The main objective of these studies is to understand how spinal input through pain pathways and chronic opioid administration interact to heighten pain sensitivity, increase opioid requirements and worsen functional outcomes after injury. The goal of the first aim is to determine how opioid exposure and surgical trauma work individually and together to regulate spinal Bdnf expression, and to define the functional consequences of that expression. We hypothesize that surgical incision and chronic opioid administration in mice converge to cause the up- regulation of Bdnf in spinal cord neurons thus enhancing the severity of opioid maladaptations and pain. Pain-related signaling involving the activation of spinal NK1 receptors by the primary afferent neurotransmitter substance P (SP) and opioid effects involving -opioid receptors (-OR) will be examined specifically using knockout mice and selective pharmacological agents. Expression studies measuring Bdnf mRNA and protein will be integrated with immunohistochemical approaches carefully identifying the cellular source(s) of BDNF in our mouse model. Additional pharmacological studies will examine the functional roles of the high affinity TrkB and low affinity p75 receptors in mediating BDNF's effects in spinal cord tissue. We expect to observe that neuronal up-regulation of Bdnf and activation of TrkB receptors contribute to multiple indices of pain after incision, functional changes in gait, tolerance and OIH. The goal of the second aim is to determine the role of epigenetics in the regulation of spinal Bdnf expression after opioid exposure, surgical trauma and the combination of circumstances. We hypothesize that the pain-related neurotransmitter SP and morphine individually and together enhance the neuronal expression of Bdnf in spinal cord dorsal horn neurons in vivo via an epigenetic mechanism. Chromatin immunoprecipitation (ChIP) assays will be used to follow changes in the association of acetylated H3K9 and/or H4K12 histone protein with the Bdnf promoter. Immunohistochemical studies will identify the cell types within the dorsal horn of the spinal cord where histone acetylation and Bdnf up-regulation are occurring. Pharmacological studies will be used to demonstrate the functional role of alterations in histone acetylation with respect to Bdnf up-regulation and worsened opioid maladaptations and pain after incision. Additional mechanism-oriented studies will examine the role of the pCREB-CBP/p300 pathway in mediating the effects of SP and morphine on histone acetylation. We expect to find that the SP and morphine pathways converge on the phosphorylation of CREB thus activating CBP/p300-mediated histone acetylation at the H3K9 or H4K12 positions near the Bdnf promoter. The proposed studies pursue the novel hypothesis that epigenetic mechanisms are responsible for clinically important adverse interactions between pain and the chronic use of opioids. In doing so we expect to identify new approaches to the treatment of pain and methods to enhance the effectiveness of our most powerful but problematic class of pain relievers. The team assembled to pursue these aims possesses expertise in all required technical aspects of the project and has the scientific and clinical experience to place the anticipated results in thei proper perspective.